Method of treating alopecia

ABSTRACT

A method of enhancing hair growth or treating alopecia in a subject uses topically administered estrogen receptor antagonists. Pharmaceutical formulations comprising estrogen receptor antagonists are described.

This application is a continuation of Ser. No. 08/604,448, filed on Feb.21, 1996, now U.S. Pat. No. 5,965,551.

This invention was made with Government support under grant CA46637 fromthe National Cancer Institute and grant ES07046 from the NationalInstitute for Environmental Health Sciences. The Government has certainrights to this invention.

FIELD OF THE INVENTION

This invention relates to methods of treating hair loss and promotinghair growth, and more particularly to a method of using estrogenreceptor antagonists to promote hair growth in a subject in need of suchtreatment.

BACKGROUND OF THE INVENTION

Alopecia (baldness) is a deficiency of hair, either normal or abnormal,and is primarily a cosmetic problem in humans, although the negativepsychological impact of hair loss is well known. See C. H. Mortimer etal., Clin. Exp. Dermatol. 9, 342-350 (1984). Dermatologists recognizemany different types of alopecia, with androgenic alopecia being themost common cause of hair loss in both men and women. As this type ofhair loss is more common and more severe in males, it is typicallyreferred to as “male pattern baldness.” However, it is thought thatandrogenic alopecia affects more that one third of individuals of eithersex who have a strong family history of hair loss. See W. F. Bergfield,Clin. Dermatol. 6, 102-107 (1988).

One traditional treatment for alopecia is the method of hairtransplantation. Typically, this method involves transplanting plugs ofnatural hair from areas of the scalp where hair is growing to baldareas. This procedure is costly, time-consuming, painful, and meets withonly limited success.

Another common treatment for hair loss is the application of a chemicalor drug for the purpose of stimulating hair growth. For example, U.S.Pat. No. 5,177,061 to Pickart proposes the topical application ofglycyl-L-histidyl-L-lycine:copper(II) (GHL-Cu) and its derivatives topromote hair growth in warm-blooded animals (applicant specificallyintends the disclosure of this and all other patent references citedherein to be incorporated herein by reference in their entirety). U.S.Pat. No. 4,832,946 to Green proposes a composition for topicalapplication to mammalian hair or skin, comprising an amount of thecell-free supernatant from a culture of dermal papilla fibroblasts,which is said to increase hair growth in the rat. U.S. Pat. No.5,358,714 to Green proposes the use of diacylglycerol activators ofprotein kinase C in order to increase or maintain hair growth inmammals, while U.S. Pat. No. 5,068,315 to Buultjens et al. proposes theapplication of purified hair growth regulating peptides (HGRP) tostimulate hair growth. It has also been suggested that retinoids,substituted pyrimidines, and immunosuppressants be used as possibletreatments for hair loss, although methods utilizing these compoundshave not been entirely successful in producing a reliable and safemethod of inducing hair growth. See G. Bazzano et al., J. Invest.Dermatol. 101 (1 Supplement), 138S-142S (1993); H. Jiang et al., J.Invest. Dermatol. 104(4), 523-525 (1995).

In recent years, the topical application of minoxidil has been awidely-used method for treating androgenic alopecia. See A. R.Zapacosta, N. Eng. J. Med. 303, 1480-81 (1980). U.S. Pat. No. 4,139,619to Chidsey, proposes a topical composition of minoxidil and relatediminopyrimidines to stimulate the conversion of vellus hair to terminalhair and increase the rate of growth of terminal hair. However, despiteits popularity, minoxidil has not performed in a completely satisfactoryfashion in promoting hair growth in all target populations.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of treating alopeciain a subject in need thereof. The method comprises topically applying tothe skin of the subject an estrogen receptor antagonist or apharmaceutically acceptable salt thereof (an “active compound”) in anamount sufficient to treat alopecia.

A more general aspect of the present invention is a method of promotinghair growth in a subject in need thereof. The method comprises topicallyapplying to the skin of the subject an active compound as given above inan amount sufficient to promote hair growth.

A second aspect of the present invention is a topical pharmaceuticalformulation comprising an active compound as given above in an amounteffective to treat alopecia or promote hair growth, in apharmaceutically acceptable topical carrier.

A third aspect of the present invention is the use of an active compoundas given above for the preparation of a medicament useful for carryingout a method as given above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photomicrograph showing the immunohistochemicallocalization of estrogen receptors in telogen hair follicle tissue frommouse skin, where immunohistochemical staining was conducted withhematoxylin counterstaining.

FIG. 1B is photomicrograph showing the immunohistochemical localizationof estrogen receptors in telogen hair follicle tissue from mouse skin,where immunohistochemical staining was conducted without hematoxylincounterstaining.

FIG. 1C is photomicrograph showing the immunohistochemical localizationof estrogen receptors in early-anagen hair follicle tissue from mouseskin, where immunohistochemical staining was conducted with hematoxylincounterstaining.

FIG. 1D is photomicrograph showing the immunohistochemical localizationof estrogen receptors in early-anagen hair follicle tissue from mouseskin, where immunohistochemical staining was conducted withoutcounterstaining.

FIG. 2 is a graph illustrating that the estrogen receptor antagonist ICI182,780 causes hair growth during the second telogen phase in mice.Closed triangles represent mice treated with 10 nmol ICI 182,780 in 200μL acetone applied topically to an area of clipped hair. Open circlesrepresent control mice treated with 200 μL acetone alone.

FIG. 3A is a photograph of a mouse treated with 200 μL of acetoneapplied topically to an area of clipped hair twice a week.

FIG. 3B is a photograph of a mouse treated with 10 nmol ICI 182,780 in200 μL acetone applied topically to an area of clipped hair twice aweek.

FIG. 4A is a photomicrograph of skin tissue from a six-week-old mousetreated with 200 μL of acetone applied topically to an area of clippedhair twice a week until seven weeks of age. The tissue was stained withhematoxylin/eosin.

FIG. 4B is a photomicrograph of skin tissue from a six-week-old mousetreated with 10 nmol ICI 182,780 in 200 μL acetone applied topically toan area of clipped hair twice a week until seven weeks of age. Thetissue was stained with hematoxylin/eosin.

FIG. 4C is a photomicrograph of skin tissue from a six-week-old mousetreated with 200 μL of acetone applied topically to an area of clippedhair twice a week until eight weeks of age. The tissue was stained withhematoxylin/eosin.

FIG. 4D is a photomicrograph of skin tissue from a six-week-old mousetreated with 10 nmol ICI 182,780 in 200 μL acetone applied topically toan area of clipped hair until eight weeks of age. The tissue was stainedwith hematoxylin/eosin.

FIG. 5 is a graph illustrating that topical 17β-estradiol blocks hairregrowth in mice. Six week old mice (5 mice per group) were clipped andtreated with topical applications of either 10 nmol 17β-estradiol in 200μL acetone (closed circles) or acetone alone (open circles), twice aweek, up to sixteen weeks of age.

FIG. 6A is a photomicrograph of skin tissue from a mouse, where 200 μLof acetone was applied topically to shaved skin twice a week for threeweeks. Tissue is stained with hematoxylin/eosin.

FIG. 6B is a photomicrograph of skin tissue from a mouse, where 10 nmol17β-estradiol in 200 μL acetone was applied topically to shaved skintwice a week for three weeks. Tissue is stained with hematoxylin/eosin.

FIG. 6C is a photomicrograph of skin tissue from a mouse, where 200 μLof acetone was applied topically to shaved skin twice a week for fiveweeks. Tissue is stained with hematoxylin/eosin.

FIG. 6D is a photomicrograph of skin tissue from a mouse, where 10 nmol17β-estradiol in 200 μL acetone was applied topically to shaved skintwice a week for five weeks. Tissue is stained with hematoxylin/eosin.

FIG. 6E is a photomicrograph of skin tissue from a mouse, where 200 μLof acetone was applied topically to shaved skin twice a week for sevenweeks. Tissue is stained with hematoxylin/eosin

FIG. 6F is a photomicrograph of skin tissue from a mouse, where 10 nmol17β-estradiol in 200 μL acetone was applied topically to shaved skintwice a week for seven weeks. Tissue is stained with hematoxylin/eosin.

DETAILED DESCRIPTION OF THE INVENTION

The method of the present invention is useful in the treatment ofalopecia in mammals, and as such may be used to promote, increase, orassist in the growth of hair. Subjects may be male or female. As usedherein, the term alopecia refers to the complete absence of hair in skinwhich typically exhibits hair growth, as well as a loss or diminution inthe amount of hair. Multiple types and causes of alopecia are recognizedin humans, including male pattern baldness, chemotherapy induced hairloss, congenital alopecia, and alopecia areata. As used herein, the term‘treating alopecia’ refers to both the treatment of skin with a totalabsence of hair growth as well as treatment of skin having reduced orpatchy hair growth.

The present invention is concerned primarily with the treatment of humansubjects but may also be employed for the treatment of other mammaliansubjects, such as dogs, cats, and sheep, for veterinary purposes (e.g.,in the treatment of hair loss due to mange or other causes, or forenhancing wool or pelt production).

Hair, or pili, are fine threadlike appendages of the skin which normallycover the entire body (with the exception of the palms of the hands andsoles of the feet, and the flexor surfaces of joints). A hair comprisesa root embedded in the hair follicle and a free portion (the stem orshaft). As used herein, hair refers to mature hair as well as the soft,downy hair known as vellus hair.

The hair bulb, or follicle, is a compact structure located in the dermislayer of the skin and is composed of three main cellular groups. Thefirst comprises a compact group of fibroblasts known as the dermalpapilla which includes a capillary system. The second group comprisesgerminative epithelial cells of the hair bulb which proliferate anddifferentiate to give rise to the mature hair shaft. The third group offibroblasts exists around the outside of the bulb in the connectivetissue sheath.

The present invention is, in general, carried out with estrogen receptorantagonists. The term “antagonist,” as used herein, refers to compoundsthat act as complete or partial inhibitors of the activity of abiological substance by specifically binding to the receptor thereof.Numerous estrogen receptor antagonists are known in the art. It isspecifically intended that both full and partial estrogen receptorantagonists are embraced by the scope of the invention, although fullestrogen receptor antagonists are preferred.

Estrogen receptor antagonists may be steroids or non-steroid compounds.In one preferred embodiment of the invention, the estrogen receptorantagonist is a steroid. Particularly preferred are 7α-alkylamideanalogs of estradiol, 11β-alkylamide analogs of estradiol and16α-halo-17β-estradiols that have pure antiestrogenic activity.

In a preferred embodiment of the invention, the estrogen receptorantagonist is a compound of Formula I:

wherein m is from 1 to 15;

n is from 1 to 5;

R₁ is hydrogen, lower alkyl, or halogen;

R₂ is oxygen or hydroxyl;

R₃ is lower alkyl or haloalkyl; and

X is

 wherein R₄ is loweralkyl.

Estrogen receptor antagonists suitable for use in the present inventioninclude, but are not limited to,7α-[9-(4,4,5,5,5,-pentafluoropentylsulfinyl)nonyl]estra-1,3,5(10)-triene-3,17β-diol(also known as ICI 182,780),N-n-butyl-N-methyl-11-(3,17β-dihydroxyestra-1,3,5(10)-triene-7α-yl)undecanamide(also known as ICI 164,384),11β-[4-[2-(dimethylaminoethoxy]phenyl]-estradiol (also known as RU39411),N-methyl-N-isopropyl-(3,17β-dihydroxy-estra-1,2,5(10)-trien-11-β-yl)-undecamide(RU 51625), its 17α-ethynyl derivative (RU 53637),[6-hydroxy-2(4-hydroxyphenyl)benzo[b]thien-3-yl][4-[3-(1-pyrrolidin-yl)ethoxy]phenyl]methanone (LY117018), tamoxifen,clomiphene, keoxifene, 3-[4-(1,2-Diphenylbut-1-enyl)phenyl]acrylic acid,and the like, including the active metabolites of these compounds. Thesecompounds may be made in accordance with known procedures which will beapparent to those skilled in the art. See, e.g., A. E. Wakeling et al.,Canc. Res, 51, 3867-3873 (1991); D. Poirier et al., J. Med. Chem, 37,1115-1125 (1994); J. Bowler et al. Steroids 54, 71-79 (1989); C.Levesque et al., J. Med. Chem, 34, 1624-1630 (1991); A. Claussner etal., J. Steroid. Biochem. Mol. Biol, 41, 609-614 (1992).

The active compounds described herein can, as noted above, be preparedin the form of their pharmaceutically acceptable salts. Pharmaceuticallyacceptable salts are salts that retain the desired biological activityof the parent compound and do not impart undesired toxicologicaleffects. Examples of such salts include (a) acid addition salts formedwith inorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; and saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; (b)salts formed from elemental anions such as chlorine, bromine, andiodine; and (c) salts derived from bases, such as ammonium salts; alkalimetal salts such as those of sodium and potassium; alkaline earth metalsalts such as those of calcium and magnesium; and salts with organicbases such as dicyclohexylamine and N-methyl-D-glucamine.

The dosage of the active compound is not particularly critical so longas it achieves the desired effect. Actual dosage will depend on factorssuch as the particular pharmaceutical carrier, the particular skinregion and condition being treated, and the general health and conditionof the subject. In one embodiment of the invention, the active compoundis topically administered to the affected skin of a subject in an amountsufficient to achieve a dose of at least 0.01 nanomoles (nmol), 0.1nmol, or 1 nmol per 2 cm by 4.5 cm skin surface area, up to a dose of100 nmol, 1,000 nmol, or 10,000 nmol or more per 2 cm by 4.5 cm skinsurface area.

Depending on the solubility of the particular formulation of activecompound administered, the daily dose may be divided among one orseveral unit dose administrations. The dose may be a single unit dose,which may, for example, be administered several times a week or from 1to 3 times a day. Treatments may continue week to week on a chronicbasis as necessary (i.e., the active agent can be administeredchronically). Administration of the active compounds may be carried outtherapeutically or prophylactically, but preferably the compounds areadministered therapeutically, either before substantial hair loss hasoccurred, or at a time when such hair loss has just begun.

Pharmaceutical compositions for use in the present method include thosesuitable for topical administration. The compositions may convenientlybe presented in unit dosage form and may be prepared by any of themethods well known in the art. The most suitable route of administrationin any given case may depend upon the anatomic location of the alopeciain the subject, the nature and severity of the condition being treated,and the particular active compound which is being used.

Pharmaceutical compositions useful in the present methods are preferablyapplied topically at the site of the affected skin. In alternativeembodiments of the invention, pharmaceutical compositions useful in thepresent invention may be administered to a subject orally, parenterally(e.g., subcutaneously, intravenously, or intramuscularly), ortransdermally (e.g., at a site other than the affected area to achievesystemic administration).

In a further aspect of the present invention, estrogen receptorantagonists may be used alone or in combination with one or moreanti-alopecics for the prophylaxis or treatment of alopecia.

In the manufacture of a medicament according to the invention (a“formulation”), active agents or the physiologically acceptable saltsthereof (the “active compound”) are typically admixed with, inter alia,an acceptable carrier. The carrier must, of course, be acceptable in thesense of being compatible with any other ingredients in the formulationand must not be deleterious to the patient. The carrier may be a solidor a liquid, or both, and is preferably formulated with the compound asa unit-dose formulation, for example, an aliquot of ointment which maycontain from 0.001% to 99% by weight of the active compound. One or moreactive compounds may be incorporated in the formulations of theinvention (e.g., the formulation may contain one or more additionalanti-alopecia agents as noted above), which formulations may be preparedby any of the well known techniques of pharmacy consisting essentiallyof admixing the components, optionally including one or more accessorytherapeutic ingredients.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include vaseline, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Additionally, the compounds of the present invention may be administeredby a liposome delivery system, as detailed in U.S. Pat. No. 4,828,837 toUster et al.

The compounds useful in the method of the present invention may beapplied the surface of the skin at any anatomical location in need oftreatment on the subject, including the scalp, pubis, face, chest, andlegs. Administration of the compounds of the present invention onto thescalp of the subject is a preferred embodiment of the invention.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, thecompound in a free-flowing form, such as a powder or granules optionallymixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Molded tablets may be made by molding, in asuitable machine, the powdered compound moistened with an inert liquidbinder. Formulations for oral administration may optionally includeenteric coatings known in the art to prevent degradation of theformulation in the stomach and provide release of the drug in the smallintestine.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.

Formulations suitable for transdermal administration (either at the sitein need of treatment or at another site to achieve systemicadministration) may be presented as discrete patches adapted to remainin intimate contact with the epidermis of the recipient for a prolongedperiod of time. Formulations suitable for transdermal administration mayalso be delivered by electrophoresis or iontophoresis (see, e.g.,Pharmaceutical Research 3, 318 (1986)) and typically take the form of anoptionally buffered aqueous solution of the active compound.

Although applicants do not wish to be bound to any particular theory ofthe instant invention, it appears that estrogen receptor antagonistscause telogen-phase hair follicles (resting follicles) to enter theanagen phase (active follicle) cycle, which results in the production ofhair fiber and hair growth. Normally, approximately 90% of scalp hairsare in the anagen phase, with less than 1% in an intermediate catogenphase and the rest in telogen. With the onset of patterned baldness,however, a successively greater proportion of hairs are in telogen,while correspondingly fewer are found in the anagen phase.

The following examples are provided to more fully illustrate the presentinvention and should not be construed as limiting thereof. As used inthe following examples, 17-β Estradiol was purchased from Sigma ChemicalCompany (St. Louis, Mo., USA); ICI 182,780 was a gift from ZenecaPharmaceuticals (Cheshire, England); link antibody, labal antibody and3,3′-diaminobenzidine tetrahydrochloride/Super Sensitive Multi-linkImmunostaining Kit were purchased from Biogenex Laboratorie (San Ramon,Calif., USA); 10× Automation Buffer was purchased from BiomedaCorporation, (Foster City, Calif., USA).

EXAMPLE 1 Animals and Estrogen or Estrogen Receptor Antagonist Treatment

Female CD-1 mice, 4, 5, or 9 weeks of age, were purchased from CharlesRiver Laboratories, Raleigh, N.C. Mice were kept in an animal facilityat least 1 week prior to use and were fed rodent chow (Agway Food,Granville Milling and Co., Creedmoor, N.C.) and water ad libitum. Themice were kept on corn cob bedding and placed on a 12 hour light/darkcycle. Female CD-1 mice were clipped on the dorsal region (approximately4-2.5 cm area) with electric clippers. The mice were then treated twiceweekly on the clipped dorsal surface with either (a) 10 nmol17-β-estradiol in 200 μl acetone or (b) acetone alone. For the studieswith the estrogen receptor antagonist, ICI 182,780 was dissolved inacetone and 200 μl was topically applied twice weekly.

EXAMPLE 2 Localization of Estrogen Receptor in Mouse Skin

Seven-week-old female CD-1 mice were killed by cervical dislocation andthe dorsal skin area was excised. The skins were fixed for 24 hours in acold 10% neutral buffered formalin, then changed to cold 70% ethanol andprocessed and embedded in paraffin. Tissue sections were cut at 5 μm andplaced on SuperFrost Plus slides for immunohistochemistry. In order tolocalize the estrogen reception in skin, the paraffin sections weredeparaffinized by two changes of xylene and rehydrated in a gradedseries of ethanol (100, 95 and 70%) followed by 1× automation buffer.The skin sections were placed in 3% H₂O₂ for 10 min to quench theendogenous peroxidase activity and then washed with 1× automationbuffer. The sections were treated with fresh trypsin of 0.15 mg/ml in 1×automation buffer for 4 min at room temperature followed by 1×automation buffer twice and incubated with fresh DNAase of 0.25 mg/ml in1× automation buffer for 3 min at room temperature followed by 1×automation buffer twice. The sections were blocked with 10% normal goatserum in 1× automation buffer for 15 min at room temperature. Afterblocking, the excessive solution was drained, and a prediluted primaryantibody (Abbott ER-ICA monoclonal antibody, Abbott Laboratories, NorthChicago, Ill.) was applied and incubated overnight at 4° C. Slides werewashed in 1× automation buffer for 5 min twice and a secondary antibody(biotinylated goat anti-rat IgG, Boehringer Mannheim Corporation,Indianapolis, Ind.) at a dilution of 1:50 in 1× automation buffer wasapplied for 60 min at room temperature. After washing with 1× automationbuffer for 5 min twice, the sections were incubated with peroxidase(HRP)-conjugated streptavidin (1:20 dilution, BioGenex, San Ramon, Fla.)for 30 min at room temperature and washed with 1× automation buffer for5 min and 0.05M Tris-HCl (pH 7.5) for 5 min. The sections were thenincubated with DAB (prepared according to the manufacturer'sinstruction) for 10 min in a dark box. Slides were rinsed with 0.05MTris-HCl buffer twice and counterstained in hematoxylin for 5 secondsfollowed by rinsing in distilled water five times. Finally, the sectionswere dehydrated in a graded series of ethanol and xylene, and thenslides were permanently mounted with Permount.

Immunohistochemical staining for the estrogen receptor in mouse skinrevealed intense and specific staining of the nuclei of cells with thedermal papilla of a telogen follicle, as shown in the counterstainedsample FIG. 1A. Non-counterstained samples (FIG. 1B) are provided tobetter demonstrate the areas and levels of estrogen receptor expression.Estrogen receptor expression within most telogen dermal papillademonstrated a polarity, as nuclei of cells within the lower half of thedermal papilli stained intensely, while very little staining wasobserved in the upper half of dermal papilla. In addition, theexpression of the estrogen receptor was hair cycle dependent as therewas a weaker staining of the dermal papilla of mid to late catagen oranagen follicles (FIGS. 1C and 1D), and no detectable staining in dermalpapilla of mid to late anagen follicles (data not shown). Very lightestrogen receptor staining was observed in cells of the outer rootsheath in the isthmus of the telogen follicle as well as in some nucleiof dermal fibroblasts.

EXAMPLE 3 Estrogen Receptor Antagonist ICI 182,780 Induces Hair Growth

Mice were treated with the estrogen receptor antagonist ICI 182,780 asprovided in Example 1. Twice-weekly treatment of clipped dorsal skin wasbegun when the mice were 6 weeks of age, with either (a) 10 nmol ICI182,780 in acetone or (b) acetone alone. As shown in FIG. 2 and FIG. 3,by 9 weeks of age, 60% of the ICI 182,780 treated mice demonstrated fullhair regrowth while no hair growth was observed in the acetone treatedmice. By 10 weeks of age all of the ICI 182,780 treated mice developed afull coat of hair while only 40% of the acetone-treated micedemonstrated full hair regrowth.

These data indicate that ICI 182,780 caused the telogen follicle toenter anagen during what should have been the second synchronizedtelogen phase. The confirm this result, skin was collected and preparedfor histological analysis at 7, 8, 9, 10 and 11 weeks. At 7 weeks of agethe ICI 182,780 treated mice demonstrated follicles that were already inearly to mid anagen, while the follicles of the acetone-treated micewere synchronized in second telogen (FIGS. 4A and 4B). By eight weeks ofage the follicles of the ICI 182,780 treated mice demonstrated folliclesthat were in mid to late anagen while the hair follicles inacetone-treated mice were in telogen (FIGS. 4C and 4D). These resultsindicate that the estrogen receptor antagonist ICI 182,780 initiates thetransition of a telogen follicle into anagen. At eleven weeks of age,the follicles of mice treated with ICI 182,780 entered telogen, whilecontrol mice were in mid to late anagen (data not shown), indicatingthat ICI 182,780 does not prolong anagen.

EXAMPLE 4 17-β Estradiol Inhibits Hair Growth

In order to determine if 17-β-estradiol could influence hair growth, sixweek old CD-1 female mice were clipped on the dorsal surface withelectric clippers and then treated twice weekly from the 6th week of ageto the 16th week of age with topical applications of either (a) 10 nmol17-β-estradiol or (b) acetone vehicle alone. Treatment with17-β-estradiol had a potent inhibitory effect on dorsal hair regrowth.As shown in FIG. 5, 100% of the acetone-treated mice demonstrated fullhair regrowth by 13 weeks of age. In contrast, mice treated with17-β-estradiol did not demonstrate any full hair regrowth by 16 weeks ofage. Some of the 17-β-estradiol-treated mice did demonstrate a partialpatchy hair regrowth that involved less than 10% of the total clippedarea. At 12 weeks of age, 20% of the 17-β-estradiol-treated micedemonstrated partial patchy hair regrowth and by 15 weeks 40% of the17-β-estradiol-treated mice demonstrated such partial patchy hairregrowth (data not shown).

The above results demonstrate that 17-β-estradiol treatment just beforeand during the 2nd telogen phase potently blocked hair growth. In orderto determine if 17-β-estradiol could block hair growth in pre-existinganagen follicles, 10 week old mice in the anagen phase of the hair cyclewere treated twice weekly with 17-β-estradiol applied topically toclipped dorsal skin. Within two weeks it was apparent that the17-β-estradiol-treated mice were no longer growing hair while all theacetone mice developed a full coat of hair (data not shown). Treatmentwith 17-β-estradiol was continued for 10 weeks and during this period nohair growth was apparent. The results of this experiment demonstratethat 17-β-estradiol can also block hair growth in mouse skin treatedduring the 3rd synchronous anagen phase.

EXAMPLE 6 Effect of 17-β-estradiol on the Hair Follicle Cycle

To determine the effect of 17-β-estradiol on the hair follicle cycle,mice were treated with 17-β-estradiol or acetone alone twice weekly fromthe 4th week of age to the 15th week of age. Every week, histologicalanalysis of the skin from 3 mice from each group was conducted todetermine the phase of the hair cycle. Representative skin histologysections from acetone- and 17-β-estradiol-treated mice at 7, 9, and 11weeks of age are shown in FIGS. 6A-6F. As expected, at 7 weeks of ageall follicles were in telogen in both the 17-β-estradiol and theacetone-treated mice. By 9 weeks of age, the hair follicles of theacetone control mice entered anagen while the hair follicles of the17-β-estradiol mice remained in telogen. By 11 weeks of age, the hairfollicles of the acetone treated mice were all in mid to late anagenwhile the hair follicles of the 17-β-estradiol-treated mice remained intelogen. At the termination of the experiment, when the mice were 15weeks of age, the hair follicles of the 17-β-estradiol-treated mice werestill arrested in telogen (data not shown). Collectively these datademonstrate that the topical application of 17-β-estradiol prevents hairre-growth by arresting the hair follicle in the telogen phase of thehair cycle.

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. The invention is defined bythe following claims, with equivalents of the claims to be includedtherein.

That which is claimed is:
 1. A method of treating alopecia in a subjectin need thereof comprising topically applying to the skin of saidsubject an estrogen receptor antagonist or a pharmaceutically acceptablesalt thereof, wherein said estrogen receptor antagonist is a steroid, inan amount sufficient to treat alopecia.
 2. A method according to claim1, wherein said subject is a female subject.
 3. A method according toclaim 1, wherein said subject is a male subject.
 4. A method accordingto claim 1, wherein said estrogen receptor antagonist is applied to thescalp of a subject.
 5. A method according to claim 1, wherein said skincontains telogen-phase hair follicles.
 6. A method according to claim 1,wherein said estrogen receptor antagonist is selected from the groupconsisting of 7α-[9-(4, 4, 5, 5, 5,-pentafluoropentylsulfinyl)nonyl]estra-1,3,5(10)-triene-3,17β-diol,N-n-butyl-N-methyl-11-(3,17β-dihydroxyestra-1,3,5(10)-triene-7α-yl)undecanamide,11β-[4-[2-(dimethylaminoethoxy]phenyl]-estradiol, andN-methyl-N-isopropyl-(3,17β-dihydroxy-estra-1,2,5(10)-trien-11-β-yl)-undecamide.7. A method of promoting hair growth in a subject in need thereofcomprising topically applying to the skin of said subject a compoundselected from the group consisting of an estrogen receptor antagonist ora pharmaceutically acceptable salt thereof, wherein said estrogenreceptor antagonist is a steroid, in an amount sufficient to promotehair growth.
 8. A method according to claim 7, wherein said subject isafflicted with hair loss, and said compound is administered in an amounteffective to treat hair loss.